Material and methods for inhibiting bacterial cell wall biosynthesis

ABSTRACT

The subject invention pertains to materials and methods for inhibiting bacterial growth. Specifically provided are compounds which control bacteria by interfering with cell wall synthesis.

BACKGROUND OF THE INVENTION

Despite the existence of many useful antibiotics, bacterial infectionsremain a major problem affecting human and animal health, agriculture,and industrial processes. The continued emergence of resistant bacteriaheightens the need for the identification of new and effectiveantibacterial agents. The most advantageous of the antibacterial agentsare those which can be used to selectively control bacteria withoutposing any health hazards for humans or animals. The identification ofsuch agents is fraught with difficulties and uncertainties due in partto the many biochemical similarities between all living organisms. Theidentification of antibacterial agents remains an empirical processrequiring extensive effort and the investment of substantial resources.

Bacteria in general synthesize a variety of extracellular glycans whichprovide structural integrity and a protective "wall" to the cell.Inhibition of the biosynthesis of this "wall" has been a favoredapproach in the design of drugs for the treatment of infectious diseasebecause animal cells do not synthesize these walls. Most of the widelyused and clinically effective antibiotics, such as the penicillins(cephalosporins) and vancomycin, function because they inhibit bacterialcell wall biosynthesis. The metabolic targets of each of these drugs areenzymes involved in the polymerization of the glycan chain and thecross-linking of the glycan chains. The biochemical pathway involved incell wall synthesis is shown in FIG. 1.

One component of importance in cell wall biosynthesis is a C₅₅isoprenoid lipid called undecaprenyl phosphate (C₅₅ -P). This lipid actsas a catalyst in gathering and transferring carbohydrate residues to thegrowing cell wall. The synthesis of a repeating saccharide unit ofpeptidoglycan occurs on the C₅₅ isoprenoid lipid at the cytoplasmic faceto the inner plasma membrane. This saccharide unit, while linked to thelipid, is translocated to the paraplasmic surface of the membrane wherethese sugars are transferred to the growing peptidoglycan.

The translocation of the C₅₅ isoprenoid lipid back to the cytosoliccompartment may be an energy dependant process. In an alternate pathway,the C₅₅ isoprenoid lipid is hydrolyzed to free undecaprenol (C₅₅ OH),which is translocated in an energy independent or low energy process tothe cytosolic side of the membrane. At the cytosolic membrane surface,undecaprenol is then phosphorylated by a ATP-dependant undecaprenolkinase to regenerate the lipid, undecaprenyl phosphate, necessary forthe next saccharide translocation process. The undecaprenol kinaseenzyme is encoded by the gene known as bac-A which has been described byCain et al. Cain, Brian D., Peter J. Norton, Willis Eubanks, Harry S.Nick, Charles M. Allen (1993) "Amplification of the bacA Gene ConfersBacitracin Resistance to Escherichia coli" J.Bacteriology175(12):3784-3789!.

Although de novo biosynthesis provides the bulk of the initial pool ofundecaprenyl phosphate, two alternative mechanisms are involved inmaintaining an adequate pool size of C55-P for cell growth: 1)regeneration of undecaprenyl phosphate from the hydrolysis ofundecaprenyl phosphate following saccharide transfer to the growingglycan; and 2) phosphorylation of free undecaprenol. The phosphorylationof free undecaprenol has previously been described as a "salvage"pathway. Although the enzyme responsible for the phosphorylation of freeundecaprenol was crystallized over 20 years ago, there is no informationpublished about its mechanism of action and little is known about itsmetabolic function.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods for killingbacteria and/or inhibiting their growth. More specifically, the subjectinvention concerns materials and methods which can be used to disruptthe processes by which the bacterial cell wall is established andmaintained. In a specific embodiment of the subject invention, bacterialgrowth is inhibited by the application of a compound which interfereswith a pathway critical for cell wall biosynthesis. Specifically, thesubject invention provides compounds which interfere with the bacterialcell's ability to maintain sufficient amounts of the C₅₅ isoprenoidlipid known as undecaprenyl phosphate (C₅₅ -P).

In a particularly preferred embodiment, the compounds useful accordingto the subject invention interfere with the bacterial cell's ability toregenerate C₅₅ -P after the C₅₅ -P has participated in thetransportation of a saccharide unit (XY) across the plasma membrane fromthe cytosol to the periplasmic space. In accordance with the subjectinvention it has been determined that the regeneration of C₅₅ -Pproceeds, at least in part, through the initial hydrolysis ofperiplasmic C₅₅ -P to C₅₅ OH followed by transport of the C₅₅ OH acrossthe plasma membrane into the cytosol. The cytosolic C₅₅ OH is thenconverted to C₅₅ -P through the action of a phosphorylating kinase knownas the undecaprenol kinase enzyme. The subject invention involves thedisruption of the conversion of C₅₅ OH to C₅₅ -P. The disruption of thisstep in the cell wall biosynthetic pathway has been found toadvantageously result in the inhibition of bacterial growth.

In a specific embodiment, the subject invention provides substratemimics which can be, for example, lipid ethers which compete with C₅₅ OHfor the undecaprenol kinase enzyme thereby reducing the rate ofconversion of C₅₅ OH to C₅₅ -P. Typically, the substrate mimics of thesubject invention would not have a terminal hydroxyl group. Instead, anether group or other group which cannot be enzymatically converted bythe kinase is present on the substrate mimic. In a preferred embodiment,the ether is a methyl or ethyl ether, but other ethers could also beused. By disrupting the normal metabolism of C₅₅ -P according to thesubject invention it is possible to achieve a cessation or significantinhibition of cell wall synthesis. Consequently, bacterial growth isinhibited.

The bacteria inhibitor compounds of the present invention can be usedfor the preparation of pharmaceutical compositions which contain aneffective amount of the active compounds, preferably in admixture with asignificant amount of inorganic or organic, solid or liquidpharmaceutically acceptable excipients. The invention also relates tosuch pharmaceutical compositions and to the preparation and use thereof.

The pharmaceutical compositions of this invention are suitable forparenteral, e.g. intravenous or intramuscular, administration, and, ascircumstances may require, also for oral administration or topicalapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the metabolic pathway involved in bacterial cell wallbiosynthesis. In a preferred embodiment, the compounds of the subjectinvention disrupt the kinase-mediated conversion of C₅₅ OH to C₅₅ -P.The disruption of this conversion reduces the amount of C₅₅ -P availablefor transporting saccharides (XY) across the plasma membrane into theperiplasmic space.

FIG. 2 shows the inhibition of bacterial growth resulting from theadministration to E. coli of farnesol which is a C₁₅ alcohol with threedouble bonds. The figure shows little inhibition of bacterial growth.

FIG. 3 shows the inhibition of bacterial growth resulting from theadministration to E. coli of farnesyl methyl ether. The figure showssignificant inhibition of bacterial growth.

FIG. 4 shows significant inhibition of E. coli growth resulting from theadministration to E. coli of bacitracin--a well-known antibiotic.

FIG. 5 shows the effect on E. coli growth resulting from theadministration of undecaprenyl methyl ether.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention provides materials and methods useful in theinhibition of bacterial growth. As used herein, reference to"inhibition" of bacterial growth refers to a reduction or prevention ofbacterial multiplication (bacteriostatic) as well as to actual killingof bacteria (bacteriocidal). The inhibition of bacterial growth achievedaccording to the subject invention is accomplished through theadministration of compounds which interfere with a bacterial cell'sability to synthesize the cell wall. The inhibition of cell wallbiosynthesis is accomplished according to the subject invention byinterfering with the cell's supply of the C₅₅ isoprenoid lipid known asundecaprenyl phosphate (C₅₅ -P). This lipid is a critical component ofthe metabolic pathway by which saccharides are transported across theplasma membrane into the periplasmic space for ultimate incorporationinto the cell wall. The metabolic pathway for cell wall biosynthesis isdepicted in FIG. 1.

In accordance with the subject invention, a bacterial cell's supply ofC₅₅ -P can be reduced by disrupting the enzymatic reaction whichconverts C₅₅ OH to C₅₅ -P. This conversion is facilitated through theactivity of a kinase known as the undecaprenol kinase enzyme. Accordingto the subject invention the ability of the undecaprenol enzyme tocatalyze the reaction which produces C₅₅ -P is decreased byadministration of inhibitor compounds.

In a specific embodiment, the subject invention provides substratemimics which can be, for example, lipid ethers which compete with C₅₅ OHfor the undecaprenol kinase enzyme thereby reducing the rate ofconversion of C₅₅ OH to C₅₅ -P. Typically, the substrate mimics of thesubject invention would not have a terminal hydroxyl group. Instead, anether group or other group which cannot be enzymatically converted bythe kinase is present on the substrate mimic. In a preferred embodiment,the ether is a methyl or ethyl ether, but other ethers could also beused. By disrupting the normal metabolism of C₅₅ -P according to thesubject invention it is possible to achieve a cessation or significantinhibition of cell wall synthesis. Consequently, bacterial growth isinhibited.

In a particularly preferred embodiment of the subject invention, theantibacterial compounds have carbon chains of less than C₅₅ therebyincreasing the water solubility of these compounds. A person skilled inthe art having the benefit of the instant disclosure can identifycompounds which have carbon chains of sufficient length to disrupt theenzymatic activity of the undecaprenol kinase enzyme while maintaining adesired degree of water solubility. These compounds are preferablyunsaturated to best mimic the native C₅₅ OH molecule. However, thedegree of unsaturation as well as the stereochemistry of the inhibitorcompound can be modified so long as the ability to disrupt enzymaticactivity is retained. It should also be noted that any negative aspectsof low water solubility can be minimized or eliminated through variousformulation modifications and/or appropriate selection of the mode ofadministration. For example, the water solubility of these lipidcompounds can be enhanced through the use of cyclodextrins which enhancewater solubility of the lipid compounds without hindering thebioavailability of those compounds. In another embodiment, the inhibitorcompounds of the subject invention can be encapsulated within a liposomeor other delivery vehicle. Liposome and cyctodextrin technologies, andother such technologies useful for enhancing water solubility, are wellknown and easily practiced by those skilled in the art and can bereadily applied in conjunction with the teachings provided herein.

Thus, in a preferred embodiment, the subject invention pertains to theinhibition of the activity of the undecaprenol kinase enzyme. It hasbeen determined that the undecaprenol kinase enzyme is an importantenzyme for bacterial extracellular glycan biosynthesis. The enzymeproduces the lipid carbohydrate carrier, undecaprenyl phosphate, whichis required by bacteria for their survival. The inhibition of theundecaprenol kinase enzyme inhibits the synthesis of extracellularglycans and therefore, bacterial cell growth.

The compounds of the subject invention can be used to control a varietyof pathogenic bacteria including, for example, M. tuberculosis, thecause of tuberculosis. Mycobacteria in general are difficult to killbecause of their resistance to most common antibiotics and theprevalence of drug-resistant strains increases the danger oftuberculosis. This resistance has been attributed to an unusual cellwall structure and the low permeability of mycobacteria to mostantibiotics. Some of the most effective antibiotics against M.tuberculosis are hydrophobic compounds, that are able to pass thishydrophobic barrier. The hydrophobic nature of the isoprenol ethers ofthe subject invention make these compounds particularly advantageous asinhibitors of this bacterium. Other bacteria which can be controlledinclude Staphylococcus pathogens as well as other bacterial pathogens.Also, the compounds of the subject invention can be used to disinfectsurfaces such as laboratory workbenches.

The active compounds of the subject invention can be used, for example,in the form of injectable compositions, e.g. for intravenousadministration, or for infusion solutions. Such solutions are preferablyisotonic aqueous solutions or suspensions which can be prepared prior touse, e.g. from lyophilished preparations which contain the activeingredient alone or together with a carrier, e.g. mannitol.Pharmaceutical compositions for oral administration may be sterilizedand can contain adjuvants, e.g., preservatives, stabilizers, wettingagents and/or emulsifiers, solubilisers, salts for regulating theosmotic pressure, resorption promoters and/or buffers. Thepharmaceutical compositions of this invention which, if desired, maycontain further useful pharmacological substances, e.g. other activeingredients, can contain about 0.1 to 100%, preferably about 1 to 100%,of active ingredient.

The compounds of the present invention can be presented foradministration to humans and animals in unit dosage forms, such assterile parenteral solutions or suspensions, containing suitablequantities of active compound. Unit dosage forms may also be as tablets,capsules, pills, powders, granules, oral solutions or suspensions, andwater-in-oil emulsions containing suitable quantities of compounds.

For oral administration, either solid or fluid unit dosage forms can beprepared. For preparing solid composition such as tablets, the unitdosage is mixed with conventional ingredients such as talc, magnesiumstearate dicalcum phosphate, magnesium aluminum silicate, calciumsulfate, starch, lactose, acacia, methylcellulose, and functionalsimilar materials as pharmaceutical diluents or carriers. Capsules areprepared by mixing the compound with an inert pharmaceutical diluent andfilling the mixture into a hard gelatin capsule of appropriate size.Soft gelatin capsules are prepared by machine encapsulation of a slurryof the compound with an acceptable vegetable oil, light liquidpetrolatum or other inert oil.

Fluid unit dosage forms for oral administration such as syrups, elixirs,and suspensions can be prepared. The water-soluble forms can bedissolved in an aqueous vehicle together with sugar, aromatic flavoringagents and preservatives to form a syrup. An elixir is prepared by usinga hydroalcoholic (ethanol) vehicle with suitable sweeteners such assugar and saccharin together with an aromatic flavoring agent.

Suspensions can be prepared with an aqueous vehicle with the aid of asuspending agent such as acacia, tragacanth, metylcellulose and thelike.

For parenteral administration fluid unit dosage forms are preparedutilizing the active compounds and a sterile vehicle. The compound,depending on the vehicle and concentration used, can be either suspendedor dissolved in the vehicle. In preparing solutions the compound can bedissolved in water for injection and filter sterilized before fillinginto a suitable vial or ampoule and sealing. Advantageously, adjuvantssuch as a local anesthetic, preservative and buffering agents can bedissolved in the vehicle. To enhance the stability, the compositions canbe frozen after filling into the vial and the water removed undervacuum. The dry lyophilized powder is then sealed in the vial and anaccompanying vial of water for injection is supplied to reconstitute theliquid prior to use. Parenteral suspensions can be prepared insubstantially the same oral suspensional manner except that the compoundis suspended in the vehicle instead of being dissolved. The compound canbe sterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of thecompound.

The term "unit dosage form," as used in the specification, refers tophysically discrete units suitable as unitary dosages for human subjectsand animals, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical diluent, carrier orvehicle. The specification for the novel unit dosage forms of thisinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular effect to beachieved and (b) the limitations inherent in the art of compounding suchan active material for use in human and animals, as disclosed in detailin this specification, these being features of the present invention.Examples of suitable unit dosage forms in accord with this invention areampoules and vials, as well as tablets, capsules, pills, powder packets,wafers, granules, cachets, teaspoonfuls, tablespoonfuls and dropperfuls,segregated multiples of the foregoing, and other forms as hereindescribed.

An effective quantity of the compound is employed in treatment. Thedosage of the compound for treatment depends on many factors that arewell known to those skilled in the art. They include, for example, theroute of administration and the potency of the particular compound. Adosage schedule for humans of from about 500 to about 5000 mg ofcompound in a single dose, administered parenterally are effective fortreating bacterial infections. When initial dosages at the lower end ofthe above range are employed, the mammal's progress can be monitored anddosages on subsequent days increased in the event that the patient oranimal response is deemed by the attending physician or veterinarian tobe absent or insufficient. The systemic toxicity of compounds of thisinvention can be carefully evaluated and subsequent dosages determinedby evaluating the benefits of the drug in relationship to any such toxicmanifestations.

For parenteral administration the compounds may be administered asinjectable dosages of a solution or suspension of the compound in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid such as water and oils with or without theaddition of a surfactant and other pharmaceutically acceptableadjuvants. Illustrative of oils which can be employed in thesepreparations are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil and mineral oil. Ingeneral, water, saline, aqueous dextrose and related sugar solutions,ethanol and glycol such as propylene glycol or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

The compounds can be administered in the form of a depot injection orimplant preparation which may be formulated in such a manner as topermit a sustained release of the active ingredient. The activeingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers or synthetic silicones, for example, Silastic, silicone rubbermanufactured by the Dow-Corning Corporation.

The pharmaceutical compositions can be prepared by conventionaldissolving or lyophilizing methods described textbooks of pharmacology.

The compounds described herein are useful antibiotic agents in animals.The term "animals" is intended to include inter alia mammals, such asmice, rats, rabbits, dogs, cats, cows, horses and primates includingman. Also encompassed within the term animals are both fish and fowl.The term "fowl" is intended to include male or female birds of any kind,but is primarily intended to encompass poultry which are commerciallyraised for eggs or meat.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Inhibition of Bacterial Growth

In one embodiment, the subject invention provides inhibitors of theundecaprenyl kinase enzyme. Specifically, ethers of isoprenyl alcoholswere tested as inhibitors of bacterial growth. E. coli cells were grownfor 18 hours on an appropriate growth medium in the presence of thevarious test compounds. Cells were then harvested and the amount ofprotein present was measured using an optical density determination. Forexample, farnesyl methyl ether was tested to determine its ability toinhibit bacterial growth. The results of inhibition studies showedclearly that the farnesol methyl ether was an effective inhibitor of thegrowth of E. coli at concentrations comparable to those found withbacitracin to inhibit bacterial growth. The parent alcohol, farnesol wasineffective in inhibiting bacterial growth over a similar concentrationrange. The results of these experiments ar shown in FIGS. 2-5.

The antibacterial use of other compounds which inhibit the ability ofthe undecaprenol kinase enzyme to convert C₅₅ OH to C₅₅ -P are withinthe subject invention.

EXAMPLE 2 Formulation and Administration

The compounds of the subject invention are useful for variousnon-therapeutic and therapeutic purposes. It is apparent from thetesting that the compounds of the invention are effective forcontrolling bacterial growth.

Therapeutic application of the compounds and compositions comprisingthem can be accomplished by any suitable therapeutic method andtechnique presently or prospectively known to those skilled in the art.

As would be appreciated by those skilled in the art, the dosageadministration will be dependent upon the identity of the infection, thetype of host involved, its age, weight, health, kind of concurrenttreatment, if any, frequency of treatment, and therapeutic ratio.

The compounds of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive compound(s) is combinedwith a suitable carrier in order to facilitate effective administrationof the composition.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

We claim:
 1. A method for inhibiting bacterial growth which comprisesadministering to said bacteria an effective amount of a compound whichinterferes with the enzymatic conversion of undecaprenol to undecaprenylphosphate.
 2. The method, according to claim 1, wherein said compoundinterferes with said enzymatic conversion by acting as a substratemimic.
 3. The method, according to claim 2, wherein said compound is alipid compound having from about 10 to about 60 carbons.
 4. The method,according to claim 1, wherein said compound is an ether.
 5. The method,according to claim 3, wherein said compound is a methyl ether.
 6. Themethod, according to claim 5, wherein said compound has between about 15and about 35 carbons.
 7. The method, according to claim 6, wherein saidcompound has about 15 carbons.
 8. The method, according to claim 7,wherein said compound is farnesyl methyl ether.
 9. The method, accordingto claim 1, wherein said method is used to treat a bacterial infectionin a mammal.
 10. The method, according to claim 9, wherein said mammalis a human.